Cells continuously experience genome damage that, if not properly attended to, can cause developmental defects, premature aging, and increased cancer predisposition. DNA damage checkpoints defend against these consequences of genomic instability by regulating cell cycle transitions, apoptosis, and DNA repair. In mammals, the checkpoint protein Hus1 is a critical component of a pathway that responds to replication stress and a variety of DNA lesions. The Hus1- dependent pathway is essential for embryonic development, and consequently many of its biological functions in animals are not well-defined. The long-term goals of the research described in this proposal are to understand how the Hus1-dependent checkpoint pathway protects genomic integrity and to determine how defects in this mechanism affect tumorigenesis and physiological DNA damage responses. The proposed studies will advance these important objectives using a unique collection of targeted modifications at the mouse Hus1 genomic locus. Aim one addresses how partial or complete Hus1 inactivation affects tumor development induced by activated oncogenes in order to resolve the opposing roles of Hus1 as a candidate tumor suppressor and a factor required for cancer cell survival and proliferation. In Aim Two, the requirements for Hus1 during in vivo DNA damage responses will be determined by measuring the tissue-specific genotoxic stress responses of mice with reduced Hus1 expression, in part to gain new insights into how checkpoint function impacts sensitivity to DNA damaging anti-cancer therapies. These analyses of the physiological consequences of checkpoint dysfunction in the first two aims will be complemented by the investigation of Hus1 molecular mechanisms in Aim Three, in an effort to define the Hus1 functions that are essential for the survival of genotoxic stress and to establish how Hus1 sequence variations affect genome maintenance. Together, the proposed studies will define how an essential checkpoint mechanism functions during tumorigenesis and tissue-specific DNA damage responses, and will provide insights into the biomedical implications of checkpoint dysfunction stemming from spontaneous mutations, natural polymorphisms, or pharmacological inhibition.